Heterocycle amide isosteres: An approach to overcoming resistance for HIV-1 integrase strand transfer inhibitors.

A series of heterocyclic pyrimidinedione-based HIV-1 integrase inhibitors was prepared and screened for activity against purified integrase enzyme and/or viruses modified with the following mutations within integrase: Q148R, Q148H/G140S and N155H. These are mutations that result in resistance to the first generation integrase inhibitors raltegravir and elvitegravir. Based on consideration of drug-target interactions, an approach was undertaken to replace the amide moiety of the first generation pyrimidinedione inhibitor with azole heterocycles that could retain potency against these key resistance mutations. An imidazole moiety was found to be the optimal amide substitute and the observed activity was rationalized with the use of calculated properties and modeling. Rat pharmacokinetic (PK) studies of the lead imidazole compounds demonstrated moderate clearance and moderate exposure.

Design, synthesis and SAR study of bridged tricyclic pyrimidinone carboxamides as HIV-1 integrase inhibitors.

The design, synthesis and structure-activity relationships associated with a series of bridged tricyclic pyrimidinone carboxamides as potent inhibitors of HIV-1 integrase strand transfer are described. Structural modifications to these molecules were made in order to examine the effect on potency towards wild-type and clinically-relevant resistant viruses. The [3.2.2]-bridged tricyclic system was identified as an advantageous chemotype, with representatives exhibiting excellent antiviral activity against both wild-type viruses and the G140S/Q148H resistant virus that arises in response to therapy with raltegravir and elvitegravir.

The discovery and preclinical evaluation of BMS-707035, a potent HIV-1 integrase strand transfer inhibitor.

BMS-707035 is an HIV-1 integrase strand transfer inhibitor (INSTI) discovered by systematic optimization of N-methylpyrimidinone carboxamides guided by structure-activity relationships (SARs) and the single crystal X-ray structure of compound 10. It was rationalized that the unexpectedly advantageous profiles of N-methylpyrimidinone carboxamides with a saturated C2-substitutent may be due, in part, to the geometric relationship between the C2-substituent and the pyrimidinone core. The single crystal X-ray structure of 10 provided support for this reasoning and guided the design of a spirocyclic series 12 which led to discovery of the morpholino-fused pyrimidinone series 13. Several carboxamides derived from this bicyclic scaffold displayed improved antiviral activity and pharmacokinetic profiles when compared with corresponding spirocyclic analogs. Based on the excellent antiviral activity, preclinical profiles and acceptable in vitro and in vivo toxicity profiles, 13a (BMS-707035) was selected for advancement into phase I clinical trials.

Improvement in aqueous solubility achieved via small molecular changes.

Overcoming poor solubility is a significant issue in drug discovery. The most common solution is to replace carbon atoms with polar heteroatoms such as N and O or by attaching a solubilizing appendage. This approach can lead to other issues such as poor activity and PK or the increased risk for toxicity. However, there are more subtle structural changes which can be employed that lead to an increase in solubility. These include, excising hydrophobic groups which do not efficiently contribute to binding, modifying stereo- and regiochemistry, increasing or decreasing the degree of unsaturation or adding small hydrophobic groups such as fluorine or methyl.

Low-event-rate meta-analyses of clinical trials: implementing good practices.

Meta-analysis of clinical trials is a methodology to summarize information from a collection of trials about an intervention, in order to make informed inferences about that intervention. Random effects allow the target population outcomes to vary among trials. Since meta-analysis is often an important element in helping shape public health policy, society depends on biostatisticians to help ensure that the methodology is sound. Yet when meta-analysis involves randomized binomial trials with low event rates, the overwhelming majority of publications use methods currently not intended for such data. This statistical practice issue must be addressed. Proper methods exist, but they are rarely applied. This tutorial is devoted to estimating a well-defined overall relative risk, via a patient-weighted random-effects method. We show what goes wrong with methods based on 'inverse-variance' weights, which are almost universally used. To illustrate similarities and differences, we contrast our methods, inverse-variance methods, and the published results (usually inverse-variance) for 18 meta-analyses from 13 Journal of the American Medical Association articles. We also consider the 2007 case of rosiglitazone (Avandia), where important public health issues were at stake, involving patient cardiovascular risk. The most widely used method would have reached a different conclusion. © 2016 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd.

Synthesis and evaluation of C2-carbon-linked heterocyclic-5-hydroxy-6-oxo-dihydropyrimidine-4-carboxamides as HIV-1 integrase inhibitors.

Integration of viral DNA into the host cell genome is an obligatory process for successful replication of HIV-1. Integrase catalyzes the insertion of viral DNA into the target DNA and is a validated target for drug discovery. Herein, we report the synthesis, antiviral activity and pharmacokinetic profiles of several C2-carbon-linked heterocyclic pyrimidinone-4-carboxamides that inhibit the strand transfer step of the integration process.

Preclinical characterization of ABI-H2158, an HBV core inhibitor with dual mechanisms of action.

The HBV core protein plays an integral role in multiple steps of the HBV lifecycle. Consequently, HBV core inhibitors interrupt multiple steps of the replication cycle, including blocking pgRNA encapsidation and prematurely disassembling existing nucleocapsids, thereby preventing them from transporting relaxed circular (rcDNA) to the nucleus for conversion to covalently closed circular DNA (cccDNA). ABI-H2158 is an HBV core inhibitor that advanced into Phase 2 clinical trials for the treatment of chronic hepatitis B virus infection (cHBV) but was discontinued due to hepatotoxicity. Here, the potency, selectivity, and mechanisms of action of ABI-H2158 were evaluated using a variety of cell-based assays. Antiviral activity was measured by quantifying intracellular or secreted HBV DNA, RNA, and antigens. ABI-H2158 inhibited HBV replication by blocking pgRNA encapsidation in induced HepAD38 cells (EC50 = 22 nM) and had similar potency in HBV-infected HepG2-NTCP cells (EC50 = 27 nM) and primary human hepatocytes (PHH) (EC50 = 41 nM). ABI-H2158 is a pan-genotypic HBV inhibitor, with EC50s ranging from 7.1 to 22 nM across HBV genotypes A-E. ABI-H2158 also potently blocked the formation of cccDNA in de novo HBV infections with EC50s of ∼200 nM in HepG2-NTCP and PHH assays. These results indicate ABI-H2158 has dual mechanisms of action, inhibiting both early and late steps of the HBV replication cycle.

Discovery and Preclinical Profiling of GSK3839919, a Potent HIV-1 Allosteric Integrase Inhibitor.

Allosteric HIV-1 integrase inhibitors (ALLINIs) have been of interest recently because of their novel mechanism of action. Strategic modifications to the C5 moiety of a class of 4-(4,4-dimethylpiperidinyl)-2,6-dimethylpyridinyl ALLINIs led to the identification of a tetrahydroisoquinoline heterocycle as a suitable spacer element to project the distal hydrophobic aryl ring. Subsequent optimization of the aryl substitutions identified 12 as an ALLINI with single-digit nanomolar inhibitory potency and low clearance across preclinical species. In preclinical toxicology studies with 12 in rats, lipid hepatocellular vacuolation was observed. Removal of the C6 methyl group resulted in GSK3839919 (22), which exhibited a reduced incidence and severity of lipid vacuolation in both in vitro assays and in vivo studies while maintaining the potency and pharmacokinetic (PK) properties of the prototype. The virology, PK, and toxicology profiles of 22 are discussed.

Solid phase synthesis of novel pyrrolidinedione analogs as potent HIV-1 integrase inhibitors.

A novel series of HIV-1 integrase inhibitors were identified from a 100 member (4R(1) x 5R(2) x 5R(3)) library of pyrrolidinedione amides. A solid-phase route was developed which facilitates the simultaneous variation at R(1), R(2), and R(3) of the pyrrolidinedione scaffold. The resulting library samples were assayed for HIV-1 integrase activity and analyzed to determine the R(1), R(2), and R(3) reagent contributions towards the activity.

Discovery and Optimization of Novel Pyrazolopyrimidines as Potent and Orally Bioavailable Allosteric HIV-1 Integrase Inhibitors.

The standard of care for HIV-1 infection, highly active antiretroviral therapy (HAART), combines two or more drugs from at least two classes. Even with the success of HAART, new drugs with novel mechanisms are needed to combat viral resistance, improve adherence, and mitigate toxicities. Active site inhibitors of HIV-1 integrase are clinically validated for the treatment of HIV-1 infection. Here we describe allosteric inhibitors of HIV-1 integrase that bind to the LEDGF/p75 interaction site and disrupt the structure of the integrase multimer that is required for the HIV-1 maturation. A series of pyrazolopyrimidine-based inhibitors was developed with a vector in the 2-position that was optimized by structure-guided compound design. This resulted in the discovery of pyrazolopyrimidine 3, which was optimized at the 2- and 7-positions to afford 26 and 29 as potent allosteric inhibitors of HIV-1 integrase that exhibited low nanomolar antiviral potency in cell culture and encouraging PK properties.

5,6,7,8-Tetrahydro-1,6-naphthyridine Derivatives as Potent HIV-1-Integrase-Allosteric-Site Inhibitors.

A series of 5,6,7,8-tetrahydro-1,6-naphthyridine derivatives targeting the allosteric lens-epithelium-derived-growth-factor-p75 (LEDGF/p75)-binding site on HIV-1 integrase, an attractive target for antiviral chemotherapy, was prepared and screened for activity against HIV-1 infection in cell culture. Small molecules that bind within the LEDGF/p75-binding site promote aberrant multimerization of the integrase enzyme and are of significant interest as HIV-1-replication inhibitors. Structure-activity-relationship studies and rat pharmacokinetic studies of lead compounds are presented.

The terminal (catalytic) adenosine of the HIV LTR controls the kinetics of binding and dissociation of HIV integrase strand transfer inhibitors.

Specific HIV integrase strand transfer inhibitors are thought to bind to the integrase active site, positioned to coordinate with two catalytic magnesium atoms in a pocket flanked by the end of the viral LTR. A structural role for the 3' terminus of the viral LTR in the inhibitor-bound state has not previously been examined. This study describes the kinetics of binding of a specific strand transfer inhibitor to integrase variants assembled with systematic changes to the terminal 3' adenosine. Kinetic experiments are consistent with a two-step binding model in which there are different functions for the terminal adenine base and the terminal deoxyribose sugar. Adenine seems to act as a "shield" which retards the rate of inhibitor association with the integrase active site, possibly by acting as an internal competitive inhibitor. The terminal deoxyribose is responsible for retarding the rate of inhibitor dissociation, either by sterically blocking inhibitor egress or by a direct interaction with the bound inhibitor. These findings further our understanding of the details of the inhibitor binding site of specific strand transfer inhibitors.

Overexpression of 9-cis-Epoxycarotenoid Dioxygenase Cisgene in Grapevine Increases Drought Tolerance and Results in Pleiotropic Effects.

9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme involved in the biosynthesis of abscisic acid (ABA), which is associated with drought tolerance in plants. An osmotic-inducible VaNCED1 gene was isolated from a drought-resistant cultivar of Vitis amurensis and constitutively overexpressed in a drought-sensitive cultivar of Vitis vinifera. Transgenic plants showed significantly improved drought tolerance, including a higher growth rate and better drought resistant under drought conditions, compared to those of wild-type (WT) plants. After water was withheld for 50 days, the upper leaves of transgenic plants remained green, whereas most leaves of WT plants turned yellow and fell. Besides the increase in ABA content, overexpression of VaNCED1 induced the production of jasmonic acid (JA) and accumulation of JA biosynthesis-related genes, including allene oxide cyclase (AOC) and 12-oxophytodienoate reductase (OPR3). Moreover, transgenic plants possessed advantageous physiological indices, including lower leaf stomatal density, lower photosynthesis rate, and lower accumulation of proline and superoxide dismutase (SOD), compared to those of WT plants, indicating increased resistance to drought stress. Quantitative real time polymerase chain reaction (RT-qPCR) analysis revealed that overexpression of VaNCED1 enhanced the expression of drought-responsive genes, such as ABA-responsive element1 (ABRE1), ABRE binding factors 2 (ABF2), plasma membrane intrinsic proteins 2 (PIP2), C-repeat/DRE-Binding Factor 4 (VvCBF4) and ABA-insensitive 5 (ABI5). Although the development of transgenic plants was delayed by 4 months than WT plants, because of seed dormancy and abnormal seedlings, the surviving transgenic plants provided a solid method for protection of woody plants from drought stress.

Confirmed and presumptive cervical vertebral compressive myelopathy in older horses: a retrospective study (1992-2004).

BACKGROUND: Cervical vertebral compressive myelopathy (CVCM) is a common cause of myelopathy in horses aged 6 months to 4 years. Little information is available regarding the types of lesions, treatment, and outcomes in horses with CVCM that are > or =4 years old. ANIMALS: Twenty-two affected horses (10 with a confirmed diagnosis of CVCM and 12 presumptive cases) and 210 contemporaneous control horses. METHODS: Horses > or =4 years old that were diagnosed with CVCM between January 1992 and January 2004 were identified from medical records at Texas A&M University and the University of Florida. Data analyzed included history, signalment, neurologic examination findings, lesion location, treatment, and outcome. Signalment was also recorded in a population of contemporaneous controls. RESULTS: Horses identified had a median age of 8.4 years, and there was a greater percentage of male horses among the cases than among the controls. The most common breeds represented were warmblood (n=6) and quarter horse (n=5) types; warmbloods were significantly (P < .05) overrepresented relative to control horses. The caudal cervical vertebral column was the most common site of CVCM lesions, and the C5-C6 (4/9) and C6-C7 (3/9) articulations were most often identified as abnormal via myelography. The most common lesions seen with radiography and myelography were articular process osteophytes. Of the 22 affected horses, 8 were euthanized and a diagnosis of CVCM was confirmed by necropsy for all; 5 of 8 of these horses had spinal cord compression caused, entirely or in part, by articular process osteophytes. Medical management was the therapy chosen in all horses, and administration of corticosteroids and nonsteroidal anti-inflammatory drugs resulted in improvement in the greatest number of horses. CONCLUSIONS AND CLINICAL IMPORTANCE: CVCM should be a differential diagnosis in older horses with cervical myelopathy. Articular process osteophytes are the most frequently identified cause of spinal cord compression in this group. Male horses and horses of warmblood or Tennessee Walking Horse breeds may be predisposed to this condition.

Evaluation of the association between spondylosis deformans and clinical signs of intervertebral disk disease in dogs: 172 cases (1999-2000).

OBJECTIVE: To evaluate the association between spondylosis deformans and clinical signs of intervertebral disk disease (IVDD) in dogs. DESIGN: Retrospective case series. ANIMALS: 210 dogs. PROCEDURE: Records of 172 dogs with clinical signs of IVDD and 38 dogs with other neurologic disorders were reviewed. Signalment, sites of spondylosis, severity of associated osteophytosis, type of disk herniation, and duration of signs were recorded. RESULTS: Dogs with IVDD had significantly fewer sites of involvement and lower grades of spondylosis deformans, compared with those in the non-IVDD group. When groups were adjusted for age and weight via multivariate linear regression, there were no differences in severity of osteophytosis or number of affected sites. Dogs with type II disk disease had higher numbers of affected sites and more severe changes, compared with dogs with type I disk herniation. There was no difference between groups in the rate at which IVDD was diagnosed at sites of spondylosis, compared with the rate at which IVDD was diagnosed in unaffected disk spaces. Areas of spondylosis were closer to sites of IVDD that elicited clinical signs than to randomly chosen intervertebral spaces, and distances between sites of spondylosis and sites of IVDD had a bimodal appearance. CONCLUSIONS AND CLINICAL RELEVANCE: An association may exist between radiographically apparent spondylosis and type II disk disease; type I disk disease was not associated with spondylosis. Spondylosis in radiographs of dogs with suspected type I disk disease is not clinically important. Spatial associations among sites of spondylosis and sites of IVDD may be coincidental or associated with vertebral column biomechanics.

Developments in antiviral drug design, discovery and development in 2004.

This article summarizes key aspects of progress made during 2004 toward the design, discovery and development of antiviral agents for clinical use. Important developments in the identification, characterization and clinical utility of inhibitors of human immunodeficiency virus; the hepatitis viruses, hepatitis B, hepatitis C; the herpes family of viruses, herpes simplex viruses 1 and 2, varicella zoster virus, Epstein-Barr virus and human cytomegalovirus; the respiratory viruses, influenza, respiratory syncytial virus, human metapneumovirus, picornaviruses, measles and the severe acute respiratory syndrome coronavirus; human papilloma virus; rotavirus; Ebola virus and West Nile virus, are reviewed.

Spiral colon bypass in a geriatric Vietnamese potbellied pig.

An 8-year-old potbellied pig was evaluated for anorexia, decreased fecal production, signs of depression, inappetence, and abdominal distension. During hospitalization, a tooth root impaction and abscess were diagnosed, and abdominal radiography revealed severely distended, gas-filled large and small intestines. Exploratory laparotomy revealed a stricture of the proximal centripetal loop of the spiral colon and megacolon of the proximal portion of the spiral colon and cecum. A side-to-side spiral colon anastomosis was performed to create a partial bypass of the spiral colon. The success of this procedure suggests that spiral colon bypass is a treatment option for spiral colon stricture formation in potbellied pigs. Spiral colon stricture formation should be considered as a differential diagnosis in geriatric potbellied pigs that are anorectic, have abdominal distension, and have decreased fecal production.

External hydrocephalus in two cats.

External hydrocephalus describes an accumulation of cerebrospinal fluid (CSF) between the cerebral hemispheres and the overlying arachnoid membrane, rather than within the lateral ventricles. Two young cats with encephalopathic signs were diagnosed with external hydrocephalus, one via magnetic resonance imaging and one via computed tomography. Both cats had abnormally large, broad heads, with no evidence of open fontanelles. A surgical shunt was placed in each cat to divert the accumulated CSF within the cranial cavity to the peritoneal space. Both cats improved dramatically soon after surgical shunting was performed, and they continue to do well clinically, approximately 42 months and 8 months postoperatively, respectively.

Novel tactics for designing water-soluble molecules in drug discovery.

INTRODUCTION: Physiochemical drug properties, such as aqueous solubility are considered to be a major factor in determining the ultimate success or failure of experimental agents. Solubility is important because it determines the maximum dose which can be taken up. As the size and hydrophobicity of drug candidates has increased over the years, poor solubility has become a more prevalent issue. Recent examples from the literature show that an improved understanding of the relationship between molecular structure and solubility allows this issue to be approached using rational design. AREAS COVERED: This review provides selected examples from the recent drug discovery literature that demonstrate various tactics, which have been applied successfully towards improving drug solubility. The examples that were selected demonstrate the underlying principles behind aqueous solubility, such as hydrophobicity and crystalline stability. EXPERT OPINION: From a strategic point of view, improving the solubility of a compound should be straightforward because it can be accomplished by simply reducing hydrophobicity or crystalline stability. However, the structural elements and physical properties which control solubility also influence potency, pharmacokinetics and toxicity. Furthermore, there are practical aspects such as the quantity and quality of solubility-related data, which hamper the development of structure-solubility relationships. Given that poor aqueous solubility remains a primary issue in drug discovery, there is a continuous need for novel methods to overcome it.

Tandem ring-closing metathesis/transfer hydrogenation: practical chemoselective hydrogenation of alkenes.

An operationally simple chemoselective transfer hydrogenation of alkenes using ruthenium metathesis catalysts is presented. Of great practicality, the transfer hydrogenation reagents can be added directly to a metathesis reaction and effect hydrogenation of the product alkene in a single pot at ambient temperature without the need to seal the vessel to prevent hydrogen gas escape. The reduction is applicable to a range of alkenes and can be performed in the presence of aryl halides and benzyl groups, a notable weakness of Pd-catalyzed hydrogenations. Scope and mechanistic considerations are presented.